Further Assessment of Monkeypox Virus Infection in Gambian Pouched Rats (Cricetomys gambianus) Using In Vivo Bioluminescent Imaging.

Monkeypox is a zoonosis clinically similar to smallpox in humans. Recent evidence has shown a potential risk of increased incidence in central Africa. Despite attempts to isolate the virus from wild rodents and other small mammals, no reservoir host has been identified. In 2003, Monkeypox virus (MPXV) was accidentally introduced into the U.S. via the pet trade and was associated with the Gambian pouched rat (Cricetomys gambianus). Therefore, we investigated the potential reservoir competence of the Gambian pouched rat for MPXV by utilizing a combination of in vivo and in vitro methods. We inoculated three animals by the intradermal route and three animals by the intranasal route, with one mock-infected control for each route. Bioluminescent imaging (BLI) was used to track replicating virus in infected animals and virological assays (e.g. real time PCR, cell culture) were used to determine viral load in blood, urine, ocular, nasal, oral, and rectal swabs. Intradermal inoculation resulted in clinical signs of monkeypox infection in two of three animals. One severely ill animal was euthanized and the other affected animal recovered. In contrast, intranasal inoculation resulted in subclinical infection in all three animals. All animals, regardless of apparent or inapparent infection, shed virus in oral and nasal secretions. Additionally, BLI identified viral replication in the skin without grossly visible lesions. These results suggest that Gambian pouched rats may play an important role in transmission of the virus to humans, as they are hunted for consumption and it is possible for MPXV-infected pouched rats to shed infectious virus without displaying overt clinical signs.

Pharmacokinetic and pharmacodynamic modeling to determine the dose of ST-246 to protect against smallpox in humans.

Although smallpox has been eradicated, the United States government considers it a "material threat" and has funded the discovery and development of potential therapeutic compounds. As reported here, the human efficacious dose for one of these compounds, ST-246, was determined using efficacy studies in nonhuman primates (NHPs), together with pharmacokinetic and pharmacodynamic analysis that predicted the appropriate dose and exposure levels to provide therapeutic benefit in humans. The efficacy analysis combined the data from studies conducted at three separate facilities that evaluated treatment following infection with a closely related virus, monkeypox virus (MPXV), in a total of 96 NHPs. The effect of infection on ST-246 pharmacokinetics in NHPs was applied to humans using population pharmacokinetic models. Exposure at the selected human dose of 600 mg is more than 4-fold higher than the lowest efficacious dose in NHPs and is predicted to provide protection to more than 95% of the population.

Ecology and geography of human monkeypox case occurrences across Africa.

As ecologic niche modeling (ENM) evolves as a tool in spatial epidemiology and public health, selection of the most appropriate and informative environmental data sets becomes increasingly important. Here, we build on a previous ENM analysis of the potential distribution of human monkeypox in Africa by refining georeferencing criteria and using more-diverse environmental data to identify environmental parameters contributing to monkeypox distributional ecology. Significant environmental variables include annual precipitation, several temperature-related variables, primary productivity, evapotranspiration, soil moisture, and pH. The potential distribution identified with this set of variables was broader than that identified in previous analyses but does not include areas recently found to hold monkeypox in southern Sudan. Our results emphasize the importance of selecting the most appropriate and informative environmental data sets for ENM analyses in pathogen transmission mapping.

In vitro inhibition of monkeypox virus production and spread by Interferon-ß.

BACKGROUND: The Orthopoxvirus genus contains numerous virus species that are capable of causing disease in humans, including variola virus (the etiological agent of smallpox), monkeypox virus, cowpox virus, and vaccinia virus (the prototypical member of the genus). Monkeypox is a zoonotic disease that is endemic in the Democratic Republic of the Congo and is characterized by systemic lesion development and prominent lymphadenopathy. Like variola virus, monkeypox virus is a high priority pathogen for therapeutic development due to its potential to cause serious disease with significant health impacts after zoonotic, accidental, or deliberate introduction into a naïve population. RESULTS: The purpose of this study was to investigate the prophylactic and therapeutic potential of interferon-ß (IFN-ß) for use against monkeypox virus. We found that treatment with human IFN-ß results in a significant decrease in monkeypox virus production and spread in vitro. IFN-ß substantially inhibited monkeypox virus when introduced 6-8 h post infection, revealing its potential for use as a therapeutic. IFN-ß induced the expression of the antiviral protein MxA in infected cells, and constitutive expression of MxA was shown to inhibit monkeypox virus infection. CONCLUSIONS: Our results demonstrate the successful inhibition of monkeypox virus using human IFN-ß and suggest that IFN-ß could potentially serve as a novel safe therapeutic for human monkeypox disease.

Experimental infection of an African dormouse (Graphiurus kelleni) with monkeypox virus.

Suitable animal models are needed to study monkeypox virus (MPXV) as human monkeypox clinically resembles smallpox and MPXV is a zoonotic and potential bioterroristic agent. We have demonstrated that a species of African dormice, Graphiurus kelleni, is susceptible to a lethal infection of MPXV and that MPXV replicated in multiple organs of this species. Following intranasal administration, MPXV replicated locally in the nasal mucosa causing necrosis and hemorrhage with subsequent systemic spread to lymph nodes, spleen, liver, and other tissues where it caused severe necrosis and/or hemorrhage leading to death. The dormouse model was validated for testing prophylactic (Dryvax vaccine) and therapeutic (cidofovir) test articles against intranasal challenges with MPXV.

Qualitative assessment of risk for monkeypox associated with domestic trade in certain animal species, United States.

In 2003, US officials identified several human monkeypox cases and traced the virus exposure to infected captive prairie dogs. The virus was likely introduced through a shipment of imported African rodents, which were kept with other mammals, including prairie dogs, in a pet distribution facility in the Midwest. To prevent the further introduction and spread of the virus, federal agencies restricted the importation of African rodents and restricted the domestic trade or movement of prairie dogs and certain other rodents. In this qualitative assessment of the risk for monkeypox associated with the 2003 outbreak, we conclude that the probability of further human infection is low; the risk is further mitigated by rodent import restrictions. Were this zoonotic disease to become established domestically, the public health effects could be substantial.

Potential antiviral therapeutics for smallpox, monkeypox and other orthopoxvirus infections.

We assessed the activities of 24 different antiviral compounds against smallpox (two strains of variola major and one of variola minor), monkeypox, vaccinia and cowpox viruses by a neutral red uptake assay. To establish assay parameters, we examined viral replication and its inhibition at various times postinfection and at several multiplicities of infection. Drugs were selected to target a range of functions involved in viral replication. Eight compounds (cidofovir, cyclic HPMPC (cHPMPC), HPMPA, ribavirin, tiazofurin, carbocyclic 3-deazaadenosine, 3-deazaneplanocin A and DFBA (1-(2,4-difluorobenzyloxy)adenosine perchlorate)-a derivative of adenosine N1-oxide) inhibited the replication of all three variola strains and the other orthopoxviruses at drug concentrations within a pharmacologically achievable range. Two others (methisazone and bis-POM-PMEA) showed a lesser degree of antiviral effect, while the remainder were inactive. To examine possible naturally occurring drug resistance among a large number of variola isolates obtained from different geographical regions and at different times, we examined the sensitivity of 35 different strains of variola as well as other orthopoxviruses to a subset of three of the most active compounds: cidofovir, cHPMPC, and ribavirin. Preliminary data indicate that nearly all isolates appear to have similar drug sensitivities. These findings are currently being verified and expanded.

Monkeypox virus as a source of whitepox viruses.

Monkeypox virus cloning and isolation of the so-called 'white' clones from white pocks which this virus forms on the chorioallantoic membrane (CAM) were carried out. The isolated clones were stable and differed considerably from the parental strain. By their properties, they were identical to whitepox viruses formerly isolated from wildlife monkeys and rodents in Equatorial Africa. Besides stable 'white' clones, a number of virus cultures in the process of cloning were obtained which differed in quantitative content of virions, forming on CAM white pocks and pocks with hemorrhages. It appeared that the properties of the viral population as a whole (reaction type on rabbit skin, hemagglutination activity, etc.) depended on the rate of virions produced with different characteristics.

Human and primate poxviruses: I. Growth characteristics of cytolytic and tumor variants.

The dual potential of poxviruses to be cytolytic and tumorgenic has been extended. Yaba monkey tumor virus formed foci on monkey and human embryonic cells. Cytolytic or plaque-forming virus was isolated from Yaba tumor homogenates by selective sucrose centrifugation and passage through monkey or human embryonic cells. Monkey pox virus (MPV) was cytolytic for monkey cells or human embryonic cells, but upon passage onto young monolayers of human carcinoma cells, HeLa, produced a restricted cytopathic effect on first transfer, foci on second transfer and cytolysis after the fourth transfer. If HeLa monolayers were compact, the rapid growth precluded overt expression of cytolytic MPV. Electron micrographs of cytolytic Yaba indicated that Yaba development was similar to vaccinia and MPV but not totally organized. Growth curves of vaccinia, MPV and cytolytic Yaba were essentially identical in monkey and human embryonic cell lines.

A poxvirus of primates. I. Growth of the virus in vitro and comparison with other poxviruses.

A primate poxvirus (OrTeCa) isolated from a skin lesion in a rhesus monkey (Macaca mulatta) was studied along with four known poxviruses: vaccinia, monkeypox, swinepox, and Yaba. It grows in established lines of monkey kidney cells at a rate intermediate between that of vaccinia and monkeypox and that of swinepox and Yaba. The cytopathic effects produced by OrTeCa virus resemble those produced by swinepox virus, although, unlike swinepox, it cannot be propagated in porcine cells. Neither can OrTeCa be grown in rabbit kidney or chick embryo cells or on chick chorioallantoic membranes, all of which support the growth of vaccinia and monkeypox viruses. OrTeCa forms plaques about the same size as those of Yaba and swinepox viruses, but they appear earlier; they are morphologically similar to the plaques of vaccinia and monkeypox viruses. The thermal stability of OrTeCa is about the same as that of vaccinia and monkeypox but greater than that of Yaba virus.